bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.265025; this version posted August 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Short title: Regulation of somatic embryogenesis by OsBABY BOOM1 2 3 Title: Rice embryogenic trigger BABY BOOM1 promotes somatic embryogenesis 4 by upregulation of auxin biosynthesis genes 5 6 Imtiyaz Khanday1, 2, Christian Santos-Medellín1,3 Venkatesan Sundaresan1, 2, 4 * 7 1Department of Plant Biology, University of California, Davis, CA, USA. 8 2Innovative Genomics Institute, University of California, Berkeley, CA, USA. 9 3 Present address: Department of Plant Pathology, University of California Davis, CA, USA. 10 4Department of Plant Sciences, University of California, Davis, CA, USA. 11 12 *To whom correspondence should be addressed: 13 Dr. Venkatesan Sundaresan 14 Department of Plant Biology, University of California, Davis, CA, USA. 15 Tel: +1 530-754-9677 16 Email: [email protected] 17 18 One-sentence summary: 19 Rice BABY BOOM1 induces somatic embryogenesis from differentiated tissues by promoting 20 auxin biosynthesis through direct upregulation of YUCCA genes. 21 Author contributions: I. K. and V. S. designed experiments; I. K. performed experiments. I. K. 22 and C. S-M. analyzed the data. I. K. wrote the manuscript with inputs from V. S. 23 Funding information: 24 C.S-M. acknowledges support from the University of California Institute for Mexico 25 (UCMEXUS), Consejo Nacional de Ciencia y Tecnología (CONACYT), and Secretaría de 26 Educación Pública (México). This research was funded by grants from the National Science 27 Foundation (IOS-1547760), Innovative Genomics Institute and the U.S. Department of 28 Agriculture (USDA) Agricultural Experiment Station (CA-D-XXX-6973-H). 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.265025; this version posted August 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 29 ABSTRACT 30 Somatic embryogenesis, a powerful tool for clonal propagation and for plant transformation, 31 involves cellular reprogramming of differentiated somatic cells to acquire pluripotency. Somatic 32 embryogenesis can be induced by treating explants with plant growth regulators. However, 33 several plant species including agronomically important cereal crops remain recalcitrant to 34 dedifferentiation and transformation except from embryonic tissues. Somatic embryogenesis can 35 also be induced by ectopic expression of select embryonic factors, including in cereals by BABY 36 BOOM (BBM) transcription factors. How BBM genes bypass the need for exogenous hormones 37 is not well understood. Here, we investigated downstream targets during induction of somatic 38 embryogenesis in rice by OsBBM1 ((Oryza sativa BABY BOOM1). Transient induction of 39 OsBBM1 led to the upregulation of auxin biosynthesis OsYUCCA genes. Continued induction of 40 OsBBM1 resulted in somatic embryogenesis without the need for exogenous auxins. Genetic 41 mutant analysis of OsBBM1 downstream targets, OsYUCCA6, OsYUCCA7 and OsYUCCA9, 42 show that they are required for normal rice development including root and shoot development. 43 Somatic embryogenic potential of OsYUCCA triple mutants was highly compromised despite the 44 presence of exogenous auxin. Additionally, we show that somatic embryogenesis induction by 45 exogenous auxin in rice requires functional BBM genes. Thus, OsBBM1 mediated cellular 46 reprogramming and somatic embryogenesis likely involves increased localized auxin through 47 direct upregulation of OsYUCCA genes. This study reveals mechanistic details of how somatic 48 embryogenesis is established in differentiated tissues in rice, a monocot model and 49 agronomically important cereal crop, with the potential utility to improve regeneration from 50 tissue culture for recalcitrant plants in future. 51 Keywords: Somatic embryogenesis, pluripotency, auxin biosynthesis, dedifferentiation, 52 regeneration. 53 54 INTRODUCTION 55 Plant cells possess the remarkable property of dedifferentiation, the ability to return to a 56 meristematic state whereby they can be redirected to various developmental fates including 57 pluripotency and totipotency. This can be achieved by endogenous or exogenous stimuli such as 58 the expression of various pluripotency factors or by treatment with plant hormones and stresses 59 (Elhiti et al., 2013; Su et al., 2020). Somatic embryogenesis is a method of clonal propagation 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.265025; this version posted August 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 60 and a powerful tool for dedifferentiation and subsequently plant regeneration. Although different 61 ways of inducing somatic embryogenesis exist in dicots (Mendez-Hernandez et al., 2019), many 62 plant species including monocots and especially agronomically important cereal crops remain 63 resistant to in vitro tissues culture, except for embryonic tissues (Eudes et al., 2003). A number 64 of plant genes such as BABY BOOM (BBM) (Boutilier et al., 2002), LEAFY COTYLEDON1 65 (LEC1), LEC2 (Lotan et al., 1998; Stone et al., 2001; Wojcikowska et al., 2013), WUSCHEL 66 (WUS) (Zuo et al., 2002), FUSCA3 (FUS3) (Luerssen et al., 1998), ABA INSENSITIVE3 (ABI3) 67 (Parcy et al., 1994; Shiota et al., 1998), EMBRYOMAKER (Tsuwamoto et al., 2010), AtMYB115, 68 AtMYB118 (Wang et al., 2009), SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 69 (SERK) (Schmidt et al., 1997) and AGAMOUS-LIKE 15 (AGL15) (Harding et al., 2003) have 70 been shown to either induce or promote somatic embryogenesis in dicotyledonous plants. 71 Among plant hormones, treatment with auxin is most widely used to induce totipotency and 72 embryogenesis from differentiated plant cells (Wojcik et al., 2020). Stress conditions like 73 osmotic stress, temperature stress, heavy metal stress, ultraviolet irradiation, wounding and 74 chemical treatments have been used to promote somatic embryogenesis (Feher, 2015; Su et al., 75 2020). 76 BABY BOOM genes are members of the AINTEGUMENTA (ANT) clade of 77 the superfamily of APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription 78 factors with two AP2 domains (Dipp-Alvarez and Cruz-Ramirez, 2019). The first member of this 79 family was identified in Brassica napus microspore cultures and its ectopic expression induced 80 somatic embryogenesis on seedlings (Boutilier et al., 2002). The property of BBM genes to 81 induce somatic embryogenesis is well documented in several other dicot species (Jha and Kumar, 82 2018). While only one BBM gene has been identified in Arabidopsis, four BBM-like genes exist 83 in rice (Dipp-Alvarez and Cruz-Ramirez, 2019; Khanday et al., 2019). In Arabidopsis, BBM has 84 been shown to express in zygotic embryos (Boutilier et al., 2002; Galinha et al., 2007); however, 85 the role of AtBBM in zygotic embryogenesis has not yet been characterized, and loss-of- 86 function mutants show no phenotypic abnormalities (Galinha et al., 2007)). We previously 87 showed that BBM genes play an important role in zygotic embryogenesis in rice; specifically, 88 that paternal expression of BBM factors is essential for embryo initiation and three BBM genes, 89 OsBBM1, OsBBM2 and OsBBM3 redundantly regulate embryo development (Khanday et al., 90 2019). 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.08.24.265025; this version posted August 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 91 YUCCA (YUC) genes encode flavin-containing monooxygenase enzymes that 92 catalyze the rate-limiting step of oxidative decarboxylation of indole-3-pyruvate acid during 93 auxin biosynthesis to form indole-3-acetic acid (IAA), the predominant auxin found in plants 94 (Cao et al., 2019). The Arabidopsis genome encodes 11 YUCCA genes, whereas 14 members of 95 this family have been identified in rice (Gallavotti et al., 2008). YUC gene mediated auxin 96 biosynthesis has been shown to play diverse roles during various phases of plant development 97 and response to environmental changes (Cao et al., 2019). YUC genes have been shown to 98 regulate root and shoot development (Chen et al., 2014; Zhang et al., 2018), leaf morphogenesis 99 (Cheng et al., 2006, 2007), inflorescence development (Woodward et al., 2005; Gallavotti et al., 100 2008), floral organ development (Cheng et al., 2006), establishment of the embryonic axis 101 (Robert et al., 2013) and embryo organ initiation (Cheng et al., 2007). 102 We previously reported that ectopic expression of OsBBM1 can induce somatic 103 embryogenesis in differentiated tissues in rice (Khanday et al., 2019), thus making it a potential 104 model for inducing and understanding somatic embryogenesis in monocots. Many downstream 105 target genes regulated by AtBBM during somatic embryogenesis have been identified